Actively Controlled and Targeted Single-Molecule Probes for Cellular Imaging Recent advances in microscopic imaging techniques with single fluorescent molecules have led to superresolution information, that is, the locations and shapes of objects in cells have been determined with resolution beyond the standard diffraction limit. These methods may be collectively termed Single-Molecule Active Control Microscopy (SMACM), because single emitting molecules are used as nanometer-scale light sources, and these emitters must be actively turned on and off to be sure that only a few molecules are emitting at any given time. Photoactivatable fluorescent protein fusions have been used for SMACM, but these emitters are large and may perturb the biological system. Though some emitters such as quantum dots provide high photostability, many additional properties are simultaneously required for advanced single-molecule imaging in cells, such as ease of functionalization, control of photophysics and photochemistry, and ease of targeting to specific cellular structures. Organic synthesis can make a huge array of "small" molecules with multiple tailored functionalities, and the present application makes use of this high degree of flexibility to develop new, targeted single-molecule emitters with active control capabilities This research will attack the problem of 3-D superresolution imaging with three interconnected thrusts which combine the skills of four investigators expert in organic synthesis, single-molecule imaging, chemistry for cellular targeting, and regulatory protein localization in bacterial cells. First, organic synthesis will generate new fluorophores with "turn-on" capability, where chemical reactivity is used to generate emission only when two protofluorophores are allowed to react, or where secondary photochemical illumination creates a fluorescent molecule in situ. Secondary illumination will also be used to photoswitch molecules on and off for additional control. The utility of the turn-on concept is that fluorescence can more easily be generated only where needed;hence backgrounds are lower. The second thrust involves selective targeting of the fluorescent labels to proteins and RNA in the cell. This will be accomplished by N-terminal cysteine labeling and RNA aptamer generation, respectively. Finally, to validate and challenge the fluorophore development, the new emitters will be used at the single-molecule level to image specific subwavelength structures, both in eukaryotic and in tiny bacterial cells. The results of this research will be to greatly extend the availability of high-resolution probes for cellular imaging at the single-molecule level, thus enabling a much deeper understanding of cellular functions. By providing a large new array of controllable and targeted single-molecule emitters, the ability of the researcher to noninvasively look inside cells will be extended into the nanoscale regime of the single-molecule emitters themselves. Public Health Relevance: The understanding of biological systems is intimately connected with unraveling disease mechanisms, and to understand the operation of the cell, optical imaging has long been an essential method by virtue of its generally noninvasive character, its capacity to assess from a distance, and its ability to observe time- dependent dynamical processes. In the cell, many small molecular machines operate one at a time, therefore scientists are now routinely observing individual single molecules, one by one, to examine the behavior of each without averaging over many inequivalent copies. To observe single molecules in cells at the spatial scale of a few tens of nm, new actively controllable and targetable emitting labels are required, and this proposed research combines the skills of four investigators to design, synthesize, and optimize a large and novel class of molecules for labeling individual proteins and RNA in living cells.